The thioredoxin redox couple thioredoxin/thioredoxin reductase (TR/Trx) is a ubiquitous redox system found in both prokaryotic and eukaryotic cells. The thioredoxin system is comprised primarily of two elements: thioredoxin and thioredoxin reductase. Thioredoxins are a class of low molecular weight redox proteins characterized by a highly conserved Cys-Gly-Pro-Cys-Lys active site. The cysteine residues at the active site of thioredoxin undergo reversible oxidation-reduction catalyzed by thioredoxin reductase. Trx-1 is ubiquitously expressed with a conserved catalytic site that undergoes reversible NADPH-dependent reduction by selenocysteine-containing flavoprotein Trx-1 reductases.
The cytosolic thioredoxin redox system is composed of thioredoxin-1 and thioredoxin reductase-1 reductase, which catalyzes the NADPH-dependent reduction of thioredoxin-1. Thioredoxin reductase-1 is an important regulator of cancer cell growth and survival. Thioredoxin-1 acting with peroxiredoxin-1 is an antioxidant that scavenges H2O2. Thioredoxins are also able to reduce buried oxidized thiol residues in proteins and regulate the activity of redox-sensitive transcription factors, including p53, nuclear factor-nB, the glucocorticoid receptor, activator protein-1, hypoxia-inducible factor-1 (HIF-1), Sp1, and Nrf2. Thioredoxin-1 also binds and inhibits the activity of the apoptosis inducing proteins, apoptosis signal-regulating kinase-1 and, the tumor suppressor phosphatase and tensin homologue deleted on chromosome 10, thus inhibiting apoptosis. Thioredoxin-1 is overexpressed in many human tumors where it is associated with increased cell proliferation, decreased apoptosis, and poor patient survival. Thioredoxin reductase thus provides a target to regulate the activity of overexpressed thioredoxin-1.
Thioredoxin reductase-1 is a selenocysteine-containing flavoprotein with broad substrate specificity because of the ready accessibility of its COOH-terminal redox active site, which contains an essential selenocysteine residue. There are three thioredoxin reductase isoforms: the canonical cytoplasmic thioredoxin reductase-1, a mitochondrial thioredoxin reductase-2, and a testes-specific thioredoxin reductase/glutathione reductase. The cellular level of thioredoxin reductase-1 is subject to complex regulation. The core promoter of the thioredoxin reductase-1 gene contains several transcription factor activation sites, including those for the redox-sensitive factors Oct-1 and Sp1 as well as others. Differential splicing and alternative transcription start sites result in multiple forms of the enzyme. Post-transcriptional regulation involving a selenocysteine insertion sequence element in the 3V-untranslated region directs selenocysteine incorporation, which is necessary for enzyme activity; thus, selenium supplementation can lead to increased thioredoxin reductase-1 activity in cell culture and in selenium deficient animals. Thioredoxin reductase-1 is necessary for cell proliferation. A thioredoxin reductase-1 knockout is embryonic lethal in mice, and thioredoxin reductase-1-deficient fibroblasts derived from the thioredoxin reductase-1 (−/−) embryos do not proliferate in vitro. Furthermore, cancer cell growth is inhibited by thioredoxin reductase-1 antisense, thioredoxin reductase-1 small interfering RNA and by a mutant redox inactive thioredoxin reductase-1. There are reports that levels of thioredoxin reductase-1 are increased by epidermal growth factor and hypoxia in cancer cells, although tumors show only moderately increased levels of thioredoxin reductase-1.
The redox protein thioredoxin-1 (Trx-1) has been proven to be a rational target for anticancer therapy involved in promoting both proliferation and angiogenesis, inhibiting apoptosis, and conferring chemotherapeutic drug resistance. Trx-1 was originally studied for its ability to act as a reducing cofactor for ribonucleotide reductase, the first unique step in DNA synthesis. Thioredoxin also exerts specific redox control over a number of transcription factors to modulate their DNA binding and, thus, to regulate gene transcription. Transcription factors regulated by thioredoxin include, but are not limited to, NF-κβ, p53, TFIIIC, BZLF1, the glucocorticoid receptor, and hypoxia inducible factor 1α (HIF-1α). Trx-1 also binds in a redox-dependent manner and regulates the activity of enzymes such as apoptosis signal-regulating kinase-1 protein kinases C δ, {dot over (ε)}, ξ, and the tumor suppressor phosphatase PTEN. Trx-1 expression is increased in several human primary cancers, including, but not limited to, lung, colon, cervix, liver, pancreatic, colorectal, and squamous cell cancer. Clinically increased Trx-1 levels have been linked to aggressive tumor growth, inhibition of apoptosis, and decreased patient survival.
Regulation of the thioredoxin-thioredoxin reductase (Trx-1/TrxR) system is attracting increasing interest due to its implication in cancer, HIV-AIDS and rheumatoid arthritis along with other medical conditions. The naphthoquinone spiroketal pharmacophore of the palmarumycin family of fungal metabolites holds promising biological activity against the Trx-1/TrxR system. Embodiments of the present invention relate to various analogues of the palmarumycin family and the ability of these analogues to inhibit the thioredoxin-thioreductase system.